The 3D F-Actin Distribution in the Ex Vivo Trabecular Meshwork (TM) of Three Species with and without Dexamethasone (DEX)

Abstract

Purpose: : The actin cytoskeleton is a key cytoplasmic component of the TM cells in the outflow system because contractility is of functional significance to aqueous drainage. There have been many 2D investigations of the F-actin in cultured TM cells, but only few reports of the 3D distribution of F-actin in intact tissue, which is of particular functional relevance especially following corticosteroid exposure.

Methods: : The TM was dissected from human (20), bovine (20) and rat (12) eyes and fixed in formal saline. The tissue samples were treated with phalloidin-Alexa 488 for F-actin and propidium iodide for nuclei, and examined by confocal microscopy. Some segments of tissue were exposed to DEX for a minimum of 3 days.

Results: : The dominant pattern of F-actin staining in the TM of all species was that of abundant stress fiber bundles running parallel with the long axis of cells. The TM sub-regions (uveal meshwork, corneoscleral meshwork and juxtacanalicular connective tissue) could be distinguished. Stress fibers were more ordered with less overlap and fewer bends in human tissues. In bovine and rat tissues actin cytoskeletons were more reticular, and stress fibers were shorter and disorganized. Cross-linked actin networks (CLANs) were identified in all three species irrespective of whether they were exposed to DEX or not, but they were of greater size, organization and complexity in human tissues. DEX exposure increased CLAN incidence in all tissue preparations, and in TM cells there were substantial disorder and tangles in the stress fibers.

Conclusions: : Despite architecturally variable F-actin patterns in the TM of the 3 species, DEX produced consistent alterations characterized by an increase in CLANs and a loss of order in stress fibers. These changes might help explain some of the cell functional alterations associated with DEX exposure to the TM. Bovine and rat TMs can be suitable animal models for the investigation of DEX-induced CLAN formation and function.

Keywords: outflow: trabecular meshwork • cytoskeleton • corticosteroids